Transendothelial electronic resistance (TEER) measurements are performed across a cell layer immobilized on a microfluidic device that also enables the cell layer to interact with a flowing stream of red blood cells (RBCs). A bipolar pulsed square wave potential is applied across a monolayer of bovine pulmonary artery endothelial cells, and the resulting current response is measured and integrated. The overall impedance of the cell layer provides an indicator of cell layer integrity. After cell seeding on the device, a decrease in TEER signal from 22.3 ± 1.6 μC to 3.5 ± 0.4 μC (corresponding to a resistance of 40.9 ± 2.9 ohms•cm2 to 259.1 ± 27.4 ohms•cm2) was observed after 8 h of cell growth. Intracellular nitric oxide (NO) production by the immobilized endothelial cells that had reached confluence was 34% higher than those cells that had not reached confluence, as indicated by the integrated TEER system. Importantly, this NO production by the confluent endothelium was stimulated by ATP released from RBCs flowing under the endothelial cells. In this construct, the described microfluidic device enables both a TEER-based evaluation of cell layer integrity and molecularly communicated interactions of these cells with a flowing stream of blood components.